Stability of SARS-CoV-2 in Phosphate-Buffered Saline for Molecular Detection

LETTER

RNA viruses often require “cold chains” of transportation to prevent the breakdown of genetic material. The logistics of getting samples from the patient to a diagnostic laboratory, sometimes thousands of miles away from the original collection site, can be complex and resource intensive (1, 2). Nucleic acid degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can compromise the accuracy of molecular detection methods. It has been demonstrated that nasopharyngeal specimens containing SARS-CoV-2 can be stored in phosphate-buffered saline (PBS) as a substitute for viral transport medium (VTM) for up to 7 days (3). Here, we evaluate the stability of differing viral loads of SARS-CoV-2 over 28 days stored at room temperature, 4°C, –20°C, or –80°C.

We used the first SARS-CoV-2-positive nasopharyngeal swab detected in our laboratory for spike-in material. PBS spiked with this SARS-CoV-2 specimen was stored in quadruplicates and divided into two concentrations, namely, 5,000 to 10,000 copies/ml (high titer) and 500 to 1,000 copies/ml (low titer), as determined by droplet digital reverse transcriptase PCR (RT-PCR). Nucleic acids were isolated on the Roche MagNA Pure96 system (Basel, Switzerland). Qualitative RT-PCR (qRT-PCR) was performed on 448 samples using our CDC-based laboratory-developed test, as described previously (4, 5).

For the high concentration of SARS-CoV-2, regardless of storage conditions, 100% of samples were detected by qRT-PCR through day 28. At room temperature, median cycle threshold (C_T) values for lower titers for both N1 and N2 targets remained consistent through day 28, fluctuating less than 1 median C_T (Table 1). For lower concentrations of virus, storage at room temperature was associated with reductions of positivity beginning at day 7, and by day 28, 0% of samples were detected for N1. Storage at room temperature was the least stable of all environmental conditions tested, with 54.2% of negative PCR results.

At 4°C, there was minimal change in C_Ts over time at the higher viral concentration. For lower titers, C_Ts increased by 2.1 C_Ts for N1 and 2.6 C_Ts for N2 over the 28 days. At –20°C, lower titers of virus fluctuated slightly more, increasing by ≥3 C_Ts. Storage of SARS-CoV-2 in PBS at –20°C was the second least stable condition, accounting for 37.5% of negative PCR results. Storage at –80°C showed the greatest stability, with all samples detected throughout the 28 days and ≤1.5 median C_Ts for both N1 and N2 targets.

Here, we show that the stability of SARS-CoV-2 can be maintained at 4°C for up to a month when –80°C storage is not available (6, 7). At viral loads of >5,000 copies/ml—corresponding to >75% of positive samples recovered in our clinical lab to date—different storage temperatures did not have a substantial impact on our ability to detect SARS-CoV-2 when stored in PBS.